Ethanol production by fermentation is a well know industrial process. However increasing ethanol yields can be technically difficult. There are various factors that make it challenging for microorganisms to grow in fermentation conditions designed for increased ethanol production. For example, the fermentation medium may have higher substrate concentrations to promote ethanol production, but these conditions can have a negative impact on cell growth. Also, increased ethanol concentration and accumulation of undesirable byproducts can also be detrimental to cell health. Yeast strains have been selected for tolerance to these conditions, which can result in improved ethanol yields. In particular, the ethanol tolerant strains of the yeast Saccharomyces cerevisiae have been used in industrial settings as a workhorse microorganism for producing ethanol.
Molecular techniques have led to the identification of genes that are associated with ethanol tolerance. For example, Kajiwara (Appl Microbiol Biotechnol. 2000; 53:568-74.) reports that overexpression of the OLE1 gene which is involved in unsaturated fatty acid (UFA) synthesis resulted in higher unsaturated fatty acid levels in the cell and higher ethanol production. Other research has found that accumulation of trehalose by disruption of the trehalose-hydrolyzing enzyme, acid trehalase (ATH) (Kim et al., Appl Environ Microbiol. 1996; 62:1563-1569) or accumulation of proline L-proline by a strain carrying a PRO1 gamma-glutamyl kinase mutation (Takagi, et al., Appl Environ Microbiol. 2005; 71:8656-8662.) improves ethanol tolerance in yeast. Ergosterol is closely associated with ethanol tolerance of Saccharomyces cerevisiae (Inoue, et al., Biosci Biotechnol Biochem. 2000; 64:229-236). While advancements have been made in this area, use of genetically modified strains that demonstrate ethanol tolerance may not alone be sufficient to provide desired levels of ethanol during a fermentation process.
In addition to the genetic profile of the fermentation microorganism, the components of the fermentation medium can have a significant impact on ethanol production. In fermentation processes, a carbohydrate or carbohydrate mixture is present in the medium. Starch is a widely available and inexpensive carbohydrate source. It is available from a wide variety of plant sources such as corn, wheat, rice, barley, and the like. Many organisms are not capable of metabolizing starch directly, or else metabolize it slowly and inefficiently.
Accordingly, it is common to treat starch before feeding it into the fermentation process, in order to break it down into monosaccharides that the organism can ferment easily. Usually, starch is hydrolyzed to form a mixture containing mainly glucose (i.e., dextrose). However, the pre-treatment of a starch composition in preparation for fermentation can be expensive and labor intensive as it commonly involves the addition of purified starch-degrading enzymes to the starch material and requires additional steps prior to carrying out fermentation. Further, complete hydrolysis to glucose adds significant cost, so most commercially available glucose products tend to contain a small amount of various oligomeric polysaccharides.
A significant portion of the cost to produce starch based ethanol is the enzymes that break down the starch into fermentable sugars. Various molecular techniques have been attempted in in Saccharomyces cerevisiae to reduce or eliminate the need to add amylolytic enzymes to the fermentation medium, but these approaches have yielded varying degrees of success. A potential limiting factor affecting the commercial viability of engineered strains is the ability of Saccharomyces cerevisiae to secrete large amounts of foreign protein.